WO2021064108A1 - Adn polymérase i marine - Google Patents

Adn polymérase i marine Download PDF

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Publication number
WO2021064108A1
WO2021064108A1 PCT/EP2020/077534 EP2020077534W WO2021064108A1 WO 2021064108 A1 WO2021064108 A1 WO 2021064108A1 EP 2020077534 W EP2020077534 W EP 2020077534W WO 2021064108 A1 WO2021064108 A1 WO 2021064108A1
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dna polymerase
seq
amino acid
sequence
dna
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PCT/EP2020/077534
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English (en)
Inventor
Atle Noralf LARSEN
Yvonne PIOTROWSKI
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Universitetet I Tromsø - Norges Arktiske Universitet
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Priority to US17/765,572 priority Critical patent/US20230220449A1/en
Priority to CA3156310A priority patent/CA3156310A1/fr
Priority to EP20780728.0A priority patent/EP4041878A1/fr
Priority to JP2022520140A priority patent/JP2022550810A/ja
Priority to CN202080069145.0A priority patent/CN114761548A/zh
Publication of WO2021064108A1 publication Critical patent/WO2021064108A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the present invention relates to DNA polymerases.
  • the present invention relates to heat labile DNA polymerases of marine origin.
  • the present invention provides heat labile DNA polymerases substantially without strand-displacement activity.
  • the present invention furthermore relates to the use of said DNA polymerase in various molecular biology processes.
  • Synthetic biology is a rapidly evolving field and is heralded as a possible solution for the challenges in future bio-economy and bioenergy.
  • the ultimate vision of synthetic biology is to create new biological operating systems of cells that predictably can carry out useful tasks.
  • One of the key steps in a synthetic biology pipeline is the assembly of DNA fragments into larger functional constructs often involving multiple assemblies.
  • a current bottleneck is however the lack of a robust room -temperature method to do multiple DNA assemblies without time-consuming manual treatment steps.
  • a new DNA assembly method able to bypass the current hurdles is therefore highly desired.
  • Replication of genomic DNA is the primary function of DNA polymerases, catalysing the synthesis for polydeoxyribonucleotides from mono- deoxyribonucleoside triphosphate (dNTPs).
  • dNTPs mono- deoxyribonucleoside triphosphate
  • DNA polymerases are used in DNA synthesis, such as in various DNA amplification processes, DNA assembly processes and in synthesis of DNA molecules reading a DNA strand template of interest creating two new DNA strands that match the template.
  • DNA polymerase I - V the known DNA polymerases are commonly referred to as DNA polymerase I - V.
  • the various groups vary in fidelity of replication, thermostability, elongation rate, and proof-reading activity and efficiency.
  • Some DNA polymerases are rather simple and others more complex, such as E.coli polymerase III that consist of 20 different peptide subunits.
  • a primer an initial oligonucleotide
  • the primer can be a short or long piece of DNA or RNA which carries a free 3' -OH group, providing a double-stranded structure to the DNA polymerase by annealing to a complementary region of a template.
  • the selected DNA polymerase works along the template, extending the primer in the 5' ⁇ 3' direction.
  • RNA molecules work as natural primers in the synthesis of both the leading strand and, in particular, the lagging strand.
  • DNA polymerase III synthesize continuously the leading strand and also the Okazaki fragments on the lagging strand, leaving gaps between the synthesized fragments that are thereafter filled by DNA polymerase I.
  • DNA polymerases may also exert other enzyme activities, such as 3’-5’ exonuclease activities or strand displacement activities.
  • the role of the strand replacement activity of DNA polymerases is to remove initiator RNA or primer before ligation.
  • the 3 ’-5’ exonuclease activity of some of the DNA polymerases is important for genetic stability, correcting DNA polymerase errors, that e.g. results in mismatched base pair in the resulting DNA molecule that is then corrected by the exonuclease function of DNA polymerases.
  • DNA polymerases In order to substitute and correct a mismatched base pair, the proof reading activity of DNA polymerases must be able to remove the incorrectly introduced dNTP and the nuclease activity are therefore involved in breaking of the phosphodiester bond in the phosphate backbone of DNA molecules.
  • the ability to remove a mismatched dNTP and thus degrade DNA is utilized in various ways in in vitro molecular biology. Sequence specific DNA amplification has many applications in molecular biology, such as in determination of paternity, forensic investigations and in diagnostics. Many of the widely used DNA polymerases are stable at high temperatures, such as up to at least 70 °C, thus enabling their use in DNA detection and analysis methods, such as polymerase chain reaction (PCR) or thermocycled DNA sequencing. DNA polymerases applicable in such processes are commonly named thermostable DNA polymerases.
  • PCR is based on thermal cycling to denature template DNA, annealing of primers and extend the primers using thermostable DNA polymerases that withstands the varying temperature conditions and by amplification exponentially increase the amount of the DNA of interest.
  • Other amplification methods are isothermal, i.e. are carried out at a constant temperature.
  • strand displacement amplification cf. e.g. Walker GT. Empirical aspects of strand displacement amplification. PCR Methods Appl. 1993;3: 1-6
  • LAMP loop-mediated amplification
  • DNA polymerases useful in strand displacement amplification are commercially available, such as the EquiPhi29TM DNA Polymerase provided by ThermoFisher Scientific.
  • DNA polymerases are also used in DNA assembly processes, such as the Gibson assembly® method, described by Gibson et al. in Nature Methods, 2009, vol. 6, pp. 343-345, allowing for a single step isotherm assembly of nucleic acid molecules.
  • the method however requires that the process is performed at 50 °C.
  • a current bottleneck is however the lack of a robust room -temperature method to do multiple DNA assemblies without time-consuming manual treatment steps.
  • the products when using PCR products in DNA assembly methods, the products must be purified (subjected to clean up procedures) before they can be used in multiple DNA assembly processes. Purification step are also required if several rounds of assembling are needed.
  • a new DNA assembly method able to bypass the current hurdles is therefore highly desired.
  • thermostable DNA polymerase of marine origin isolated from Psychrobacillus sp. being active at a wide range of temperatures, including temperatures above room temperature.
  • WO20 16026574 discloses a thermolabile exonuclease originating from a cold-water environment being capable of degrading single stranded DNA, and which may be inactivated within 15-20 minutes if exposed to temperatures below 65 °C.
  • the present inventors have identified a DNA polymerase I by metagenomic analysis of marine environmental samples collected in the marine artic area around Svalbard.
  • the present isolated DNA polymerases are intrinsically heat labile which renders the enzymes specifically useful in molecular biology processes, such as in a variety of DNA amplification processes and DNA assembly processes.
  • the present DNA polymerase is rapidly and irreversible inactivated at temperatures above 25°C, such as at temperatures above about 30 °C, resulting in no need for any inactivation step before further handling of a product being subjected to the DNA polymerase of the present invention.
  • the present inventors have shown that the present DNA polymerase exert a very robust polymerase activity compared with commercially available DNA polymerases, such as the mesophilic Klenow enzyme from E. coli and the thermophilic Bst polymerase originating from Bacillus stearothermophilus .
  • the robust polymerase activity as well as the temperature lability characteristics of the present DNA polymerase makes it a very useful DNA polymerase for a wide range of DNA amplification processes, which can be performed at room- temperature and which avoids the need of an inactivation step.
  • the present DNA polymerase furthermore exerts 3’-5’ exonuclease activity, resulting in proof reading of the replicated DNA molecule.
  • the present DNA polymerase furthermore possesses a strand displacement activity, making it an attractive polymerase for strand displacement amplification processes
  • the present inventors have also synthesized modified variants of the DNA polymerase of the present invention, wherein the strand displacement activity of the DNA polymerase is sufficiently impaired or absent.
  • modified DNA polymerases of the present invention with impaired or lacking strand displacement activity is in particularly useful in recombinant cloning processes, e.g. wherein two or more double stranded nucleic acid molecules with single stranded 5’ overhang is assembled.
  • a modified DNA polymerase with impaired or lacking strand displacement activity is useful in multiple DNA assembly methods, and because of its heat liability renders it possible to work at room temperature.
  • a further advantage of the present DNA polymerase is that when used in DNA amplification or DNA assembly processes, as shown further below, no inactivation step deemed is necessary.
  • an isolated DNA polymerase or an enzymatically active fragment thereof wherein said DNA polymerase exert strand- displacement activity, 3 ’-5’ exonuclease activity, and wherein said DNA polymerase is irreversibly inactivated at temperatures above 25 °C, more preferably at temperatures above about 30 °C.
  • a DNA polymerase wherein the strand displacement activity is reduced, impaired or inactivated.
  • an isolated DNA polymerase or an enzymatically active fragment thereof comprising the amino acid sequence of SEQ ID No. 1, or comprising an amino acid sequence which is at least 60% sequence identical over the entire length of the sequence with SEQ ID No. 1.
  • an isolated DNA polymerase or an enzymatically active fragment thereof is provided, said DNA polymerase comprising the amino acid sequence of SEQ ID No. 2, or comprising an amino acid sequence which is at least 60% sequence identical over the entire length of the sequence with SEQ ID No. 2.
  • the isolated DNA polymerase or an enzymatically active fragment thereof may comprise an amino acid sequence which is at least 70% identical over the entire length of the sequence with SEQ ID No. 1 or SEQ ID No. 2, such as at least 80% sequence identical over the entire length of the sequence with SEQ ID No. 1 or SEQ ID No. 2, such as at least 90% sequence identical over the entire length of the sequence with SEQ ID No. 1 or SEQ ID No.
  • an isolated DNA polymerase or an enzymatically active fragment thereof wherein said amino acid sequence comprises at least one mutation in at least one of the amino acid regions corresponding to amino acid positions 431 - 447 and positions 519-523, the numbering being accordance with the amino acid numbering in SEQ ID NO. 2, and wherein said DNA polymerase have no strand-displacement activity.
  • said DNA polymerase or an enzymatically active fragment thereof comprises at least one mutation in amino acid positions corresponding to S449, A450, F451, and/or R521 of an amino acid sequence as set forth in SEQ ID No. 1 and SEQ ID No. 2.
  • an isolated DNA polymerase or an enzymatically active fragment thereof comprises at least one mutation in amino acid positions corresponding to S449, A450, F451, and/or R521 of an amino acid sequence as set forth in SEQ ID No. 1 and SEQ ID No. 2, and wherein the at least one mutation is a substitution to: an amino acid with a hydrophobic side chain at a position corresponding to S449, an amino acid with a negative charged side chain at a position corresponding to A450, an amino acid with a hydrophobic side chain at a position corresponding to F451, and/or an amino acid with a hydrophobic side chain at a position corresponding to R521.
  • said isolated DNA polymerase or an enzymatically active fragment thereof may according to one embodiment of the above aspect comprise an amino acid sequence wherein the amino acid in position 449 according to the numbering of SEQ ID No. 1 or SEQ ID No. 2 is selected from the group consisting of Ser, Ala, Gly, Val, Leu, lie.
  • said isolated DNA polymerase or an enzymatically active fragment thereof may according to one embodiment of the above aspect, comprises an amino acid sequence, wherein the amino acid in position 450 according to the numbering of SEQ ID No. 1 or SEQ ID No. 2 is selected from the group consisting of Ala, Gly, Val, Leu, lie, Asp, Glu, Asn, Gin.
  • said isolated DNA polymerase or an enzymatically active fragment thereof may according to one embodiment of the above aspect, comprises an amino acid sequence, wherein the amino acid in position 451 according to the numbering of SEQ ID No. 1 or SEQ ID No. 2 is selected from the group consisting of Phe, Ala, Gly, Val, Leu, IIe.
  • said isolated DNA polymerase or an enzymatically active fragment thereof may according to one embodiment of the above aspect, comprises an amino acid sequence, wherein the amino acid in position 521 according to the numbering of SEQ ID No. 1 or SEQ ID No. 2 is selected from the group consisting of Arg, Ala, Gly, Val, Leu, IIe.
  • said isolated DNA polymerase or an enzymatically active fragment thereof may according to one embodiment of the above aspect, comprises an amino acid sequence, wherein the amino acid in position 449, 450, 451 and 521 is selected from the groups consisting of provided that the amino acids in position 449 (S449), 450 (A450), 451 (F451) and 521 (R521) are not at the same time Ser, Ala, Phe and Arg, respectively.
  • said isolated DNA polymerase or an enzymatically active fragment thereof may according to one embodiment of the above aspect, comprises an amino acid sequence, wherein the amino acid in position 449, 450, 451 and 521 is selected from the groups consisting of and provided that the amino acids in position 449 (S449), 450 (A450), 451 (F451) and 521 (R521) is not at the same time Ser, Ala, Phe and Arg, respectively.
  • the isolated DNA polymerase or an enzymatically active fragment thereof is selected from a group of DNA polymerases comprising an amino acid sequence wherein
  • amino acids in position 449 and 450 are Ala and Asp, respectively.
  • amino acids in position 450 and 451 are Asp and Ala, respectively.
  • amino acids in position 449, 450 and 451 are Ala, Asp and Ala, respectively,
  • the amino acid in position 521 in SEQ ID No. 8 is Ala and wherein the numbering is according to numbering of the amino acids of SEQ ID No. 1 and wherein any of said DNA polymerases has no strand strand-displacement activity.
  • an isolated DNA polymerase or an enzymatically active fragment thereof comprising the amino acid sequence selected from the group consisting of SEQ ID No. 3, 4, 5, 6, 7 and 8, or comprising an amino acid sequence which is at least 60% such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85% such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98% or 99% sequence identity over the entire length of the sequence with SEQ ID No. 3, 4, 5, 6, 7, and 8, respectively, provided that
  • amino acids in position 449 and 450 in SEQ ID No. 5 are Ala and Asp, respectively,
  • amino acids in position 450 and 451 in SEQ ID No. 6 are Asp and Ala, respectively,
  • amino acids in position 449, 450 and 451 in SEQ ID No. 7 are Ala, Asp and Ala, respectively,
  • the present invention also provides according to any of the above aspects a DNA polymerase or an enzymatically active fragment thereof, wherein the enzyme is irreversibly inactivated at temperatures above 25 °C, such as at temperatures above 30 °C
  • composition comprising an isolated DNA polymerase or an enzymatically active fragment thereof according to any of the preceding aspects and a buffer.
  • the DNA polymerase is a large fragment DNA polymerase I lacking the N-terminal 5 ’-3 ’-exonuclease domain.
  • SEQ ID No. 1 and Seq ID No. 2 are examples of large fragment DNA polymerase sequences lacking the N-terminal 5 ’-3 ’-exonuclease domain.
  • a nucleic acid molecule is provided encoding an isolated DNA polymerase or an enzymatically active fragment thereof according to any of the above aspects.
  • the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID No. 9 or comprising a nucleic acid molecule which has at least 60% sequence identity over the entire length of the sequence of SEQ ID No. 9.
  • the nucleic acid molecule comprises a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 or comprising a nucleic acid molecule encoding an amino acid sequence which has at least 60% sequence identity over the entire length of the sequence with SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, respectively.
  • an expression vector comprising a nucleic acid molecule encoding an isolated DNA polymerase or an enzymatically active fragment thereof according to the above aspects and the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by said nucleic acid molecule.
  • Said expression vector may for example comprise a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 or comprising a nucleic acid molecule encoding an amino acid sequence which has at least 60% sequence identity over the entire length of the sequence with SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, respectively.
  • a host cell comprising one or more expression vectors as mentioned above, or one or more nucleic acid molecules mentioned above encoding the DNA polymerase of the present invention.
  • a method for preparation of a DNA polymerase or an enzymatically active fragment thereof according to the invention comprising the steps of: a) culturing a host cell comprising one or more of the recombinant expressions vectors according to according to the tenth aspect, or one or more nucleic acid molecules according to the ninth aspect under conditions suitable for the expression of the encoded DNA polymerase; b) isolating or obtaining the DNA polymerase such as a large fragment DNA polymerase from the host cell or from the culture medium or supernatant.
  • the present invention furthermore relates to the use of a DNA polymerase such as a large fragment DNA polymerase or an enzymatically active fragment of the invention in a nucleic acid amplification process, sequencing reaction, recombinant cloning process or multiple DNA assembly processes.
  • a DNA polymerase such as a large fragment DNA polymerase or an enzymatically active fragment of the invention in a nucleic acid amplification process, sequencing reaction, recombinant cloning process or multiple DNA assembly processes.
  • the DNA polymerase of the present invention is used in strand displacement amplification.
  • a fourteenth aspect a method for assembly of two or more double stranded (ds) DNA molecules is provided, said process comprising the steps of:
  • step (c) contacting the annealed molecules with a heat labile DNA polymerase such as a large fragment DNA polymerase or an enzymatically active fragment thereof according to any of aspect one to sixth, whereby the DNA polymerase fill in the gaps remaining after the annealing of DNA molecules formed in step (b), wherein said DNA polymerase have reduced, impaired or inactivated strand displacement activity.
  • a heat labile DNA polymerase such as a large fragment DNA polymerase or an enzymatically active fragment thereof according to any of aspect one to sixth
  • steps (a) - (c) of the above method may be carried out at constant temperature. According to one embodiment, said process is carried out at a temperature within the range of 20 °C to 25°C. According to another embodiment, the assembled DNA molecule of step (c) is further transferred into a suitable host cell for propagation.
  • a method is provided, wherein the overhang of the two or more DNA molecules of step (a) of the above method is provided using a 3’- 5 ’exonuclease, preferably a heat labile exonuclease.
  • a method of nucleotide polymerization is provided using a DNA polymerase such as a large fragment DNA polymerase or enzymatically active fragment thereof of the invention, said method comprising the steps of:
  • a method of amplifying a nucleic acid using a DNA polymerase such as a large fragment DNA polymerase or enzymatically active fragment thereof comprising the steps of:
  • reaction mixture comprising a DNA polymerase or enzymatically active fragment thereof according to any one aspects one to six , a template nucleic acid molecule, an oligonucleotide primer(s) which is capable of annealing to a portion of the template nucleic acid molecule acid molecule, and nucleotides;
  • Figure 1 represents the Klenow fragment (PDB code: 1D8Y), a homologous polymerase to the DNA polymerases of the present invention, illustrating the alpha helix identified by the arrow harboring the three consecutive amino acid residues S449, A450 and F451, and also showing the position of residue R521, the C- and N- terminal end.
  • PDB code: 1D8Y a homologous polymerase to the DNA polymerases of the present invention
  • Figure 2 shows the DNA and amino acid sequence of the DNA polymerase of the present invention.
  • Figure 3 shows the polymerase activity of the present large fragment DNA polymerase compared with the polymerase activity of the Klenow enzyme from E. coli and the thermophilic Bacillus stearothermophilus (Bst) polymerase.
  • Figure 4 shows the results of experiment measuring the residual activity of the present wild type large fragment DNA polymerase at 25 °C after incubation of the enzyme at various temperatures.
  • Figure 5 shows a comparison of the polymerase activity of the large fragment DNA polymerases of the present invention at 25°C, represented by the wild type (wt) DNA polymerase, the A4 SOD -mutant (SDF), S449A+F451 A-mutant , mutant (ADF), the A450D+ F451 A-mutant (SDA), the -mutant (ADA) and the R521 A-mutant.
  • Figure 6 shows a comparison of the strand displacement activity of the large fragment DNA polymerases of the present invention at 25 °C, represented by the wild type (wt) DNA polymerase, the A4 SOD -mutant (SDF), S449A+F451 A-mutant ( ) -mutant (ADF), the A450D+ F451 A-mutant (SDA), the -mutant (ADA) and the R521 A-mutant.
  • SDF wild type DNA polymerase
  • SDF S449A+F451 A-mutant
  • ADF S449A+F451 A-mutant
  • SDA A450D+ F451 A-mutant
  • ADA -mutant
  • the present inventors have as mentioned identified a novel DNA polymerase of marine origin with advantageous characteristics which makes said DNA polymerase and variants thereof useful in a number of molecular biology processes.
  • the enzyme may be used in processes carried out at room temperature, and that is it easily inactivated, such as at a temperature above 25 °C, such as above 30 °C.
  • the DNA polymerase of the invention may be used in order to provide assembly target nucleic acid molecules with 5’-3’ overhang, e.g. in order to provide a full sequence nucleic acid molecule and combine said molecule with a vector.
  • the DNA polymerase may thus be used to assembly one or more target nucleic acid molecule in a carrier or expression vector, wherein the desired nucleic acid molecule(s) and the vector has complementary 5 ’-3’ overhang.
  • the DNA polymerase of the present invention upon contacting the one or more target double stranded nucleic acid molecule and a vector of choice, both having 5’- 3’ overhangs, e.g. of a length of about 10-40 base pair, the DNA polymerase of the present invention will fill in the number of nucleotides needed in order to assembly the sequences in questions. Due to the DNA polymerases heat liability, the DNA polymerase will be inactivated within a short time but is active for a sufficient time in order to e.g. assembly the nucleic acid molecules in question.
  • the DNA polymerase of the present invention may become inactive over time at 25 °C, however it has been shown to maintain its activity for at least 60 minutes, it does not influence further process steps e.g. when used in multiple DNA assembly methods.
  • annealed DNA molecules are brought in contact with a DNA polymerase with reduced, impaired or inactivated strand displacement activity, for a period of time in the range of 5-45 minutes, such as in the range of 10-30 minutes, such as in the range of 15 - 20 minutes.
  • the enzyme of the present invention may be used in various processes carried out at room temperature.
  • room temperature is a recognized term in the art and includes temperatures in within the range of 18 °C to 25 °C.
  • a DNA polymerase or an enzymatically active fragment thereof comprising the amino acid sequence of SEQ ID No. 1 or SEQ ID NO. 2, or comprising an amino acid sequence which is at least 60% sequence identical over the entire length of the sequence with SEQ ID No. 1.
  • an enzymatically active fragment of the DNA polymerase is to be understood to mean a DNA polymerase where the activity of the polymerase is maintained, that is having the same or at least similar activity compared with a DNA polymerases having an amino acid sequence as depicted in SEQ ID No. 1-8, although one or more amino acids are removed compared with the sequences depicted in SEQ ID No. 1 -8.
  • the skilled person will acknowledge that one or more amino acid may be removed, e.g. in the C- or N-terminal end of an amino acid sequence, without affecting the activity of the protein.
  • the DNA polymerase of the present invention exerts a superior polymerase efficiency compared with known DNA polymerases.
  • the present DNA polymerase show improved polymerase activity compared with the polymerase activity of the Klenow enzyme from E. coli and the thermophilic Bacillus stearothermophilus (Bst) polymerase.
  • Bst Bacillus stearothermophilus
  • the skilled person will furthermore acknowledge that polymerase activity can be measured using a real time molecular beacon assay, such as disclosed in Summerer, Methods Mol. Biol., 2008, 429, 225- 235 or in modified form as shown in the below experimental part.
  • a DNA polymerase is provided substantially without strand-displacement activity and wherein said DNA polymerase is substantially without strand-displacement activity and furthermore is irreversibly inactivated at temperatures above 25 °C, such as temperatures above about 30 °C.
  • strand displacement activity can be measured using well known methods, such as the strand displacement activity assay described in Piotrowski et al., 2019, BMC Mol Cell Biol, 20 (31) (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688381/1.
  • substantially without strand displacement activity is to be understood to mean that the displacement activity of the DNA polymerase is impaired or absent compared with the wild type DNA polymerase the wild type DNA polymerase having an amino acid sequence according to SEQ ID No. 2.
  • a DNA polymerase having a displacement activity that is reduced to the degree of the DNA polymerases having an amino acid sequence of SEQ ID NO. 3-8 has an impaired strand displacement activity, i.e. that are substantially without strand displacement activity.
  • the present invention provides a DNA-polymerase such as an isolated large fragment DNA polymerase or an enzymatically active fragment thereof comprising an amino acid sequence of SEQ ID No. 1 or amino acid sequences that are at least 60% sequence identical over the entire length of the sequence with SEQ ID No. 1.
  • a DNA-polymerase such as an isolated large fragment DNA polymerase or an enzymatically active fragment thereof comprising an amino acid sequence of SEQ ID No. 1 or amino acid sequences that are at least 60% sequence identical over the entire length of the sequence with SEQ ID No. 1.
  • the present invention provides a DNA-polymerase such as an isolated large fragment DNA polymerase or an enzymatically active fragment thereof comprising an amino acid sequence of SEQ ID No. 2 or amino acid sequences that are at least 60% sequence identical over the entire length of the sequence with SEQ ID No. 2.
  • a DNA-polymerase such as an isolated large fragment DNA polymerase or an enzymatically active fragment thereof comprising an amino acid sequence of SEQ ID No. 2 or amino acid sequences that are at least 60% sequence identical over the entire length of the sequence with SEQ ID No. 2.
  • a DNA polymerase comprising an amino acid according to SEQ ID No. 1 or SEQ ID No. 2 and comprising at least one mutation in the regions corresponding to amino acid positions 431 - 447 and positions 519- 523, the numbering being accordance with the amino acid numbering in SEQ ID NO. 2, and wherein said DNA polymerase have no strand-displacement activity.
  • the amino acids in position 431 to 447 make up three helixes believed to be involved in the strand displacement activity of the identified marine DNA polymerase of the present invention.
  • a DNA polymerase is provided comprising an amino acid according to SEQ ID No. 1 or SEQ ID No. 2 and comprising at least one mutation in the regions corresponding to amino acid positions G447-L453 and positions G519-A523, the numbering being accordance with the amino acid numbering in SEQ ID NO. 2.
  • a DNA polymerase is provided wherein a mutation is introduced in position 449, 450, 451 and/or 521.
  • the present invention provides as examples wherein inter alia serine in position 449 is replaced by alanine, or wherein alanine in position 450 is replaced by asparagine, or wherein phenylalanine in position 451 is replaced by alanine, or wherein arginine in position 521 is replaced by alanine.
  • amino acids are grouped dependent upon the chemical characteristics of the side chain. Amino acids are commonly classified as hydrophobic or hydrophilic and/or as having polar or non-polar side chain. Substitutions of one amino acid for another having the same biochemical characteristics are commonly known as conservative substitution. The skilled person will acknowledge that conservative substitutions can be introduced into an amino acid sequence of a protein, e.g. to the enzyme according to the present invention without altering the activity of said enzyme. Such modifications will thus be expected to constitute a biologically equivalent product.
  • amino acids within the following groups are amino acids within the following groups:
  • Gin amino acids with uncharged side chains
  • a conservative amino acid substitution refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made, and thus seldom alter the three-dimensional structure of the protein, which is why the biological activity are neither altered significantly.
  • a DNA polymerase such as an isolated large fragment DNA polymerase comprising an amino acid sequence according to SEQ ID No. 1 or SEQ ID No. 2, wherein the amino acid in position 449 is selected from the group consisting of Ser, Ala, Gly, Val, Leu, lie and/or the amino acid in position 450 is selected from the group consisting to Ala, Gly, Val, Leu, lie, Asp, Glu, Asn, Gin, and/or wherein the amino acid in position 451 is selected from the group consisting of Phe, Ala, Gly, Val, Leu, lie, and/or wherein the amino acid in position 521 is selected from Arg, Ala, Gly, Val, Leu, lie, provided that the amino acids in position 449 (S449), 450 (A450), 451 (F451) and 521 (R521) is not Ser, Ala, Phe and Arg, respectively, may have the same or approximately the same polymerase activity and strand displacement activity as a DNA polyme
  • amino acids may be deleted, inserted or added without altering the activity of the DNA-polymerase.
  • Large Fragment DNA Polymerase I is a DNA polymerase enzyme that lacks the 5' to 3' exonuclease activity of intact DNA Polymerase I, but does exhibit the 5' to 3' DNA polymerase and 3' to 5' exonuclease activities.
  • An example of a well-known large fragment DNA polymerase I is the Klenow fragment.
  • the present invention provides a DNA-polymerase or an enzymatically active fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID No. 3, 4, 5, 6, 7, and 8, or comprising an amino acid sequence which is at least 60% sequence identical over the entire length of the sequence with SEQ ID No. 3, 4, 5, 6, 7, and 8, respectively.
  • a DNA polymerase comprising an amino acid sequence having at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85% such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98% or 99% sequence identity over the entire length of the sequence with an amino acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.
  • the present invention also provides a nucleic acid molecule encoding an isolated DNA polymerase or an enzymatically active fragment thereof according to the present invention.
  • a nucleic acid molecule comprising a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 or comprising an amino acid sequence which is at least 60% sequence identical over the entire length of the sequence with SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively.
  • nucleic acid molecule comprising the sequence as depicted in SEQ ID No. 9 or nucleic acid molecules which is at least 80% sequence identical over the entire length of the sequence with SEQ ID No. 9, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99% sequence identical over the entire length of the sequence with SEQ ID No. 9.
  • amino acids may be deleted, inserted or added without altering the activity of the enzyme of the present invention.
  • sequence identity a sequence having at least x% identity to a second sequence means that x% represents the number of amino acids in the first sequence which are identical to their matched amino acids of the second sequence when both sequences are optimally aligned via a global alignment, relative to the total length of the second amino acid sequence. Both sequences are optimally aligned when x is maximum. The alignment and the determination of the percentage of identity may be carried out manually or automatically. Whenever referring to sequence identity herein, it is to be understood that the comparison is made with the entire sequence depicted in SEQ ID NO. 1 - SEQ ID No. 9, respectively.
  • NCBI BLAST is another example of software used to determine amino acid sequence identity (MacWilliam et al., Nucleic Acids Res. 2013 Jul; 41 (Web Server issue): W597-W600).
  • modifications may be introduced in a nucleic acid molecule which does not alter the amino acid sequence, e.g. the substitution of a nucleotide resulting in that the triplet affected by the substitution still codes for the same amino acid.
  • the amino acid isoleucine is encoded by the triplets (DNA codons) ATT, ATC, and ATA.
  • a substitution in the third nucleotide in the isoleucine triplet ATT from T to C or A will not alter the resulting amino acid sequence.
  • Such nucleotide modifications may be introduced by techniques well known to the skilled person (e.g. site directed mutagenesis) to adapt the nucleic acid sequence to the codons preferably used by a host cell and thus to enhance the expression of the enzyme.
  • nucleic acid molecules coding polypeptides which facilitates isolation and purification can be added to the nucleotide sequences of the present invention without affecting the activity of the resulting DNA polymerase.
  • nucleic acid molecules coding signal peptide providing for secretion of the desired enzyme from a host cell may also be linked to the nucleic acid sequences of the present invention.
  • the present invention furthermore provides a composition comprising the DNA polymerase of the present invention or enzymatically active fragment thereof.
  • the composition comprising an enzyme of the present invention may comprise buffers for optimised activity of the enzyme.
  • buffers used in composition comprising an enzyme of the invention may vary and optimised according to the enzyme of choice and the process wherein the enzyme is used.
  • the enzyme of the invention is retained within the conditions commonly used in molecular biology processes such as cloning processes, DNA assembly processes and DNA amplification processes well known to the skilled person, that is e.g. in respect of type and concentration of salt(s), pH conditions, etc.
  • Tris buffer such as Tris buffer may be used, such as a Tris buffer having a pH above about 8.0, for example a pH within the range of 8.0 and 9.0. According to one aspect, the pH of the composition is within 8.5 - 9.0.
  • the composition comprises one or more salts selected from the group consisting of NaCl and KC1.
  • the composition comprises NaCl and KC1.
  • the composition comprises about 50 mM or more NaCl and about 50 mM or more KC1.
  • the DNA polymerase of the present invention and the enzymatically active fragments thereof or the nucleic acid molecule encoding them, is purified from or isolated from their natural environment or they are produced by recombinant DNA procedures well known to the skilled person.
  • Nucleic acid molecules encoding a DNA polymerase according to the present invention or encoding an enzymatically active fragment thereof may synthesized by methods well known to the skilled person or commercial suppliers well known to the skilled person, e.g. Genscript, Thermo Fisher Scientific etc.
  • nucleic acid molecule encoding the enzymes according to the present invention or encoding an enzymatically active fragment thereof may be inserted in a suitable expression vector comprising all the necessary transcriptional and translational regulatory sequences specifically adapted for directing the expression of the desired protein coding nucleic acid sequence in a suitable host cell.
  • suitable expression vectors are e.g. plasmids, cosmids, viruses or artificial yeast chromosomes (YAC’s).
  • DNA molecules to be expressed and used to prepare a DNA polymerase according to the present invention may be inserted into vectors used for propagation of the sequence of interest or for expression of the DNA polymerase encoding sequence of the invention.
  • FastCloning is an example of an applicable method for this purpose.
  • a vector such as an expression vector comprising the nucleic acid molecule encoding a DNA polymerase according to the present invention or an enzymatically active fragment thereof.
  • a vector such as an expression vector comprising a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, or amino acid sequences having at least about 60% sequence identity over the entire length of the sequence such as at least, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length of the sequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.
  • a vector comprising SEQ ID No. 9, or nucleic acid molecules which is at least 80% sequence identical over the entire length of the sequence with SEQ ID No. 9, or a sequence with 80% sequence identity over the entire length of the sequence of SEQ ID No. 9, such as at least, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length of the sequence of SEQ ID No. 9.
  • a DNA polymerase according to the present invention may be prepared using an expression vector comprising a nucleic acid molecule encoding a DNA polymerase according to the present invention, wherein said molecule is operably linked to a promotor adapted for the host cell in question.
  • a “promoter” as used herein refers to a region of DNA upstream (5 ’-prime) of a DNA coding sequence that controls and initiates transcription of the particular gene.
  • the promoter controls recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • “Operably linked” refers to a functional linkage between a promoter and a second sequence, where the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous.
  • a vector adapted for expression of recombinant proteins in bacterial host cells may comprise a promotor applicable for bacterial expression systems, such as the T7 promoter.
  • a vector according to the present invention may be isolated using standard plasmid isolation techniques well known to the skilled person, such as e.g. using a QIAprepTM Spin Miniprep kit from QiagenTM or QIAGENTM Plasmid Plus Maxi Kit.
  • bacterial host cells such as E.coli, BL21 cells or Rosetta 2 (DE3) cells (Novagen). Transformation of the expression vector may be performed by methods well known to the skilled person, e.g. using chemically competent cells.
  • the DNA polymerase of the present invention or an enzymatically active fragment thereof encoded by the expression vector in the host cell will be produced, and the resulting DNA polymerase may be collected and purified by methods well known to the skilled person.
  • the expression vector may furthermore include signal sequences for secretion of the expressed enzyme into the culture media.
  • the DNA polymerase of the present invention may be synthesized using recombinant DNA technology.
  • the DNA polymerase of the present invention is prepared using cell-free expression systems, or it may be manufactured using chemical peptide synthesis methods, e.g. by stepwise condensation reaction of the carboxyl group of one amino acid to the amino group of another in accordance with the desired sequence of amino acids.
  • a process for the preparation of a DNA polymerase of the present invention or a enzymatically active fragment thereof comprising the steps of (i) culturing a host cell comprising one or more expression vectors of the present invention suitable for the expression of the encoded DNA polymerase; and optionally (ii) isolating or obtaining the DNA polymerase from the host cell or from the culture medium (supernatant).
  • a recombinant expressed protein from a host cell or a culture medium.
  • one or more pre-treatments or clarification steps is commonly used first in order to remove large particles and biomass.
  • Nonlimiting examples of applicable pre-treatment steps are e.g. reverse osmosis, centrifugation, filtration methods and diafiltration, or a combination thereof.
  • the obtained enzyme is then commonly purified by one or more of a variety of chromatographic methods well known to the skilled person, e.g. by affinity chromatography, ion-exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography, size exclusion chromatography or other chromatography techniques, or a combination thereof.
  • an enzyme expressed by a suitable host cell may be purified using an affinity chromatography method, such as using MabSelectTM SuReTM media and a HiTrap MabSelectTM SuReTM column mounted on an FPLC chromatography system, e.g. the BioRad NGC DiscoverTM 10 Pro system fitted with a 5mm UV flow cell.
  • an affinity chromatography method such as using MabSelectTM SuReTM media and a HiTrap MabSelectTM SuReTM column mounted on an FPLC chromatography system, e.g. the BioRad NGC DiscoverTM 10 Pro system fitted with a 5mm UV flow cell.
  • the column is commonly washed one or more times with one or more applicable wash buffers, where after the protein is eluted using an applicable elution buffer.
  • the obtained enzyme may be further purified using one or more of the chromatography methods listed above.
  • the present enzyme may be used in any molecular biology process where DNA polymerases are utilized.
  • the DNA polymerase may be used in DNA amplification methods and DNA assembly processes, in particular multiple DNA assembly processes.
  • the DNA polymerase is particular advantageous to use in molecular biology processes carried out at room temperature.
  • the DNA polymerase of the invention is also useful in molecular biology processes where inactivation steps are preferably avoided.
  • the present DNA polymerase is particularly useful in methods for assembly of nucleic acid molecules based on homologue recombination, and methods adapted for assembly of a large number of nucleic acid molecules.
  • the present DNA polymerase may be used in DNA assembly process as disclosed in EP1915446B1 or in in vitro recombination methods like the one disclosed in EP1929012B1.
  • the ends to be assembled should share sequence identity ensuring that the respective overhangs of in question resulting from the exonuclease digestion step anneals (hybridize).
  • the length of the overhang is preferably of a length sufficient to hybridize specifically to complementary overhangs of the shared region of sequence identity, so as to allow hybridization of the single-stranded overhangs.
  • the DNA polymerase of the present invention is particularly suitable due to that polymerization process can be carried out at room temperature. Furthermore, as the DNA polymerase of the present invention is heat labile, and inactivated at temperatures above about 25°C, resulting in that the polymerization process is easily ceased without the use of laborious inactivation steps. The fact that the present DNA polymerase exert a proof-reading activity in the form of a 3 ’-5’ exonuclease makes it applicable in high fidelity amplification processes.
  • the DNA polymerase of the present invention having impaired or that lack strand displacement activity is in particularly useful in multiple DNA assembly processes.
  • DNA polymerase of the present invention is a large fragment DNA polymerase lacking 5 ’-3’ exonuclease domain and having impaired or lacking strand displacement activity is also particularly useful in multiple DNA assembly processes.
  • Example 1 Identification of the DNA polymerase (MG pol I) and modification thereof by site-directed mutagenesis
  • the vector pET151/D-TOPO ® containing the codon-optimized gene encoding the large fragment of the identified DNA polymerase (SEQ ID No.9) was purchased from the Invitrogen GeneArt Gene Synthesis service from Thermo Fisher Scientific.
  • Recombinant protein production of MG Pol I large fragment and its mutants was performed in Rosetta 2 (DE3) cells (Novagen ® ). The cells grew in Terrific Broth media/ampicillin (100 ⁇ g/ml) and gene expression was induced at ODeoo nm 1.0 by addition of 0.1 mM IPTG. Protein production was carried out at 15 °C overnight.
  • the pellet of a 1 ⁇ 2-l cultivation was resuspended in 50 mM HEPES pH 7.5 (at 25 °C), 500 mM NaCl, 5 % glycerol, 1 mM DTT, pH 7.5, 0.15 mg/ml lysozyme, 1 protease inhibitor tablet (CompleteTM, Mini, EDTA-free Protease Inhibitor Cocktail, Roche) and incubated on ice for 30 min. Cell disruption was performed by sonication with the VCX 750 from Sonics ® (pulse 1.0/1.0, 15 min, amplitude 25 %).
  • the soluble part of the Hise-tagged protein present after centrifugation (48384g, 45 min, 4 °C) and filtration (0 0.45 pm) was purified by immobilized Ni 2+ -affinity chromatography.
  • the protein was eluted at an imidazole concentration of 250 mM and further transferred into 50 mM HEPES, 500 mM NaCl, 10 mM MgClz, 5 % glycerol, pH 7.5 by use of a desalting column.
  • the second step was the cleavage of the tag by the TEV protease performed over night at 4 °C in 50 mM Tris pH 8.0, 0.5 mM EDTA and 1 mM DTT.
  • a second Ni 2+ -affinity chromatography has been performed in the third step by applying 50 mM HEPES, 500 mM NaCl, 5 % glycerol, 1 mM DTT, pH 7.5.
  • the final protein solution was concentrated and stored with 50 % glycerol at -20 °C for activity assays.
  • Example 3 Measuring of polymerase activity of the present enzyme.
  • the molecular beacon template consists of a 23mer loop that is connected by a GC-rich 8mer stem region (sequence is indicated in italics) and a 43mer extension. Due to the loop formation the fluorophores Dabcyl and FAM are in close proximity and thus quenched.
  • the molecular beacon substrate was produced by incubating 20 ⁇ of 10 ⁇ molecular beacon template and 15 ⁇ primer in 10 mM Tris-HCl pH 8.0, 100 mM NaCl for 5 min at 95 °C. The reaction was then let to cool down at room temperature for 2 h. The substrate solution was stored at -20 °C with a final concentration of 10 ⁇ .
  • Fifty microliter reactions consisted of 200 nM substrate and 200 ⁇ dNTP (equimolar amounts of dATP, dGTP, dCTP and dTTP).
  • the reaction further contained 5 mM MgCh in 50 mM Tris-HCl pH 8.5, 100 mM KC1, 1 mM DTT, 0.2 mg/ml BSA and 2 % glycerol.
  • the activity assay was carried out at 25 °C in black 96-well fluorescence assay plates (Corning ® ).
  • the reaction was initiated by addition of protein solution, i.e. MG pol I and its variants.
  • the increase in FAM fluorescence was measured as relative fluorescence units in appropriate time intervals by exciting at 485 nm and emission at 518 nm. The measurement was performed in a SpectraMax ® Gemini Microplate Reader (Molecular Devices).
  • the assay is based on an increase in fluorescence signal that is measured upon displacement of the quenched reporter strand. This is only achievable through strand-displacement activity of the DNA polymerase.
  • the substrate for the strand-displacement activity assay consists of a “cold” primer of 19 oligonucleotides and a reporter strand consisting of 20 oligonucleotides that is labeled with the TAMRA fluorophore [TAMRA] at its 3’ end.
  • the template strand consists of 40 oligonucleotides and is labeled with the Black Hole Quencher 2 (BHQ2) at its 5’ end.
  • BHQ2 Black Hole Quencher 2
  • the primers are annealed to the template strand leaving a one- nucleotide gap at position 20 on the template strand. Furthermore, are the labels in close proximity and thus the fluorophore TAMRA is quenched by BHQ2.
  • the TAMRA labeled oligonucleotide Upon strand-displacement activity of the DNA polymerase I the TAMRA labeled oligonucleotide is displaced from the template strand. As a consequence, the fluorophore and the quencher are no longer in close proximity and an increase in TAMRA fluorescence can be measured (excitation 525 nm, emission 598 nm).
  • the strand-displacement activity of the DNA polymerase of the present invention and its variants expressed as mRFU/min/pg has been analyzed using the above- described strand-displacement activity assay.
  • the substrate for the strand-displacement activity assay was produced by incubating 20 ⁇ of 10 ⁇ “cold” primer, 10 ⁇ reporter strand and 10 ⁇ template strand in 10 mM Tris-HCl pH 8.0, 100 mM NaCl at 95 °C for 5 min. The reaction was then let to cool down at room temperature for 2 h. The substrate solution was stored at - 20 °C with a final concentration of 10 ⁇ .
  • Fifty microliter reactions consisted of 200 nM substrate and 200 ⁇ dNTP (equimolar amounts of dATP, dGTP, dCTP and dTTP).
  • the reaction further contained 5 mM MgCl 2 in 50 mM Tris-HCl pH 8.5, 100 mM KC1, 1 mM DTT, 0.2 mg/ml BSA and 2 % glycerol.
  • the activity assay was carried out at 25 °C in black 96-well fluorescence assay plates (Corning ® ). The reaction was initiated by addition of protein solution, i.e. MG pol I and its variants.
  • TAMRA fluorescence was measured as relative fluorescence units in appropriate time intervals by exciting at 525 nm and recording emission at 598 nm. The measurement was performed in a SpectraMax ® M2 e Microplate Reader (Molecular Devices).

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Abstract

La présente invention concerne des ADN polymérases. En particulier, la présente invention concerne des ADN polymérases thermostables d'origine marine, ayant une activité de polymérase élevée, une activité de déplacement de brin et une activité exonucléase 3' à 5'. En outre, la présente invention concerne des ADN polymérases thermo-instables sensiblement sans activité de déplacement de brin.
PCT/EP2020/077534 2019-10-01 2020-10-01 Adn polymérase i marine WO2021064108A1 (fr)

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US17/765,572 US20230220449A1 (en) 2019-10-01 2020-10-01 Marine dna polymerase i
CA3156310A CA3156310A1 (fr) 2019-10-01 2020-10-01 Adn polymerase i marine
EP20780728.0A EP4041878A1 (fr) 2019-10-01 2020-10-01 Adn polymérase i marine
JP2022520140A JP2022550810A (ja) 2019-10-01 2020-10-01 海洋性ｄｎａポリメラーゼｉ
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